Long-term memory formation is accompanied by changes in neuronal architecture. Several lines of evidence suggest that the serine protease, tissue plasminogen activator (tPA), serves as a modulator of learning-related synpatic plasticity. To better understand the molecular determinants of synaptic plasticity, we propose to localize tPA in hippocampal neurons and study the activity-dependent secretion of tPA from synaptic structures. In addition, studies in this proposal addresss fundamental questions regarding the trafficking of tPA in regulated secretory granules in cultured hippocampal neurons. Fluorescence microscopy and green fluorescent protein (GFP) technology will be used to localize tPA/GFP in cultured hippocampal neurons and determine if the distribution of the secretory granules containing tPA/GFP is influenced by processes such as axonal outgrowth, dendritic maturation and synaptogenesis. The participation of tPA in activity-dependent synaptic plasticity will be evaluated by using tPA/GFP to investigate release during synaptic stimulation of hippocampal neurons. Finally, time-lapse images of the transport of regulated secretory granules containing tPA/GFP will be generated. Granule dynamics will be characterized to provide insight into the molecular events that influence the transport and secretion of proteins from regulated secretory granules.